Toxicon 124 (2016) 63e72 Contents lists available at ScienceDirect Toxicon journal homepage: www.elsevier.com/locate/toxicon Serine proteinases from Bothrops snake venom activates PI3K/Akt mediated angiogenesis Shreesha K. Bhat a, Manjunath B. Joshi a, Anwar Ullah b, Rehana Masood b, * Setlur G. Biligiri c, Raghuvir K. Arni b, Kapaettu Satyamoorthy a, a School of Life Sciences, Manipal University, Manipal, India b Multiuser Center for Biomolecular Innovation, IBILCE/UNESP, Sao~ Jose do Rio Preto, SP, Brazil c Juggat Pharma, Jagadale Industries, Bangalore, India article info abstract Article history: The discovery of rapid acting and powerful angiogenic proteins are of significant interest in the treatment Received 14 July 2016 of various human disorders associated with insufficient angiogenesis such as ischemia, menorrhagia and Received in revised form delayed wound healing. Snake venoms consist of a mixture of bioactive proteins and polypeptides and 28 October 2016 are rich sources of pharmacologically important molecules. Serine proteinases are one of the abundant Accepted 1 November 2016 proteins present in Bothrops snake venoms and possess multiple biological functions including the Available online 2 November 2016 regulation of the blood coagulation cascade. In this study, serine proteinases from Bothrops atrox (B. atrox) and Bothrops brazili (B. brazili) that modulate angiogenesis were purified and characterized. Keywords: fi Serine proteinases Molecular size exclusion chromatography, af nity chromatography followed by ion exchange chroma- Bothrops tography of the serine proteinases indicated molecular masses of around 32 kDa. Serine proteinases from Venom both the species exhibited diverse catalytic activities such as the ability to induce amidolytic, fibrino- Blood coagulation genolytic, gelatinolytic activities and also coagulated plasma with a minimal coagulation concentration of Angiogenesis 2.4 mg/mL. Serine proteinases facilitated the sprouting of human umbilical vein endothelial cells (HUVEC) Therapeutic applications in three-dimensional culture systems and induced tubule formation in monolayer culture systems. Serine proteinase stimulated Aktser473 and eNOSser1177 phosphorylation in endothelial cells and addition of PI3K inhibitor LY294002 abrogated the effects of serine proteinases on sprout formation of endothelial cells in 3D collagen gels, suggesting that serine proteinase facilitated angiogenesis was mediated by PI3K/ eNOS signaling axis. We also show in agarose plug assays using a mouse model, serine proteinases from Bothrops venoms significantly enhanced neovascularization. Our data suggests pro-angiogenic activity by the serine proteinases from B. atrox and B. brazili venom and further studies are warranted to explore the therapeutic applications. © 2016 Elsevier Ltd. All rights reserved. 1. Introduction (Gutierrez and Lomonte, 1989). Bothrops snake venoms constitute mixtures of biologically active substances, comprising proteins, Snakes belonging to the Bothrops genus are pit vipers specificto lipids, steroids, aminopolysaccharides, amines and quinines (Vyas fi Central and South America and exhibit numerous intraspeci c et al., 2013). Metalloproteinases, phospholipases A2, serine pro- differences in their morphological and ecological traits (McDiarmid teinases and C-type lectins present in Bothrops venoms have been et al., 1999; Carrasco et al., 2012). Venoms from Bothrops species demonstrated to interfere with the hemostatic system (Markland, contain highly toxic and bioactive substances and cause myonec- 1998; Matsui et al., 2000). Snake venom serine proteinases rosis, cardiovascular alterations such as hemorrhage and hypo- (SVSPs) that are present abundantly in venoms of Viperidae, Cro- volemic shock, edema, coagulation disorders and renal alterations talidae, Elapidae and Colubridae form a group of comprehensively studied toxins affecting several physiological processes (Serrano and Maroun, 2005). Serine proteinases form a part of the largest family of peptidases including trypsin family S1 of clan SA (Halfon * Corresponding author. School of Life Sciences, Manipal University, Manipal and Craik, 1998). Diverse biological functions of proteinases have 576104, India. E-mail address: [email protected] (K. Satyamoorthy). been reported such as the regulation of cytokines and signaling http://dx.doi.org/10.1016/j.toxicon.2016.11.001 0041-0101/© 2016 Elsevier Ltd. All rights reserved. 64 S.K. Bhat et al. / Toxicon 124 (2016) 63e72 receptors leading to the modulation of intracellular signaling Manipal University, Manipal, India. Briefly, endothelial cells from pathways (caspase-like enzyme activity) (Ramachandran and veins were dislodged using collagenase Type IV for 30 min, fol- Hollenberg, 2008). One of the targets of SVSPs for cleavage are lowed by washing with calcium and magnesium free phosphate mainly the proteins involved in the coagulation cascade of fibri- buffered saline (PBS pH 7.4). Cells were seeded on gelatin coated nolytic and kallikreinekinin systems which can significantly in- plates and further cultured until confluency. Endothelial cells were fluence hemostatic systems (Serrano, 2013). characterized by cell specific markers such as CD31, VE-cadherin Angiogenesis (neovascularization) is the physiological process and CD44 using flow cytometry (BD Biosciences, USA). Endothe- that involves growth of new blood vessels (Folkman, 1984). It has lial cells were cultured in Endothelial Cell Growth Medium (ECGM) long been recognized that several pathological conditions are (Promo cell GMBH, Germany) containing 5% Fetal Bovine Serum associated with the component of either excessive angiogenesis or (FBS). Fibroblast cells were isolated from skin epidermis, charac- insufficient angiogenesis (Carmeliet, 2003). Pathological angio- terized based on typical fibroblast phenotype and were cultured in genesis is responsible for more than 70 life-threatening diseases. DMEM with 10% FBS (Himedia, Mumbai, India). Hepatocarcinoma Accordingly, much effort has been devoted to identify target mol- (HepG2), human transformed embryonal kidney cells (HEK293), ecules that can be exploited for therapeutic strategies by inhibiting human transformed epithelial cells (HaCaT), Madin-Darby Canine or promoting angiogenesis (Noel et al., 2004; Ribatti and Djonov, Kidney Cells (MDCK) and neuroblastoma (IMR32) cell lines were 2011). Pathological conditions such as ischemia in ischemic heart procured from ATCC and cultured in DMEM containing 10% FBS. diseases, menorrhagia and delayed wound healing are associated Peripheral blood mononuclear cells (PBMC) were isolated from with insufficient angiogenesis. Several pre-clinical studies have healthy volunteers and cultured in RPMIþ2% fetal bovine serum. reported the use of various proteins such as vascular endothelial growth factor, platelet derived growth factor and fibroblast growth 2.3. Purification of serine proteinases from B. brazili and B. atrox factor-2 (bFGF) to induce angiogenesis for the treatment of diseases associated with the impaired angiogenesis (Said et al., 2012). Serine 2.3.1. Molecular size exclusion chromatography proteinases and other proteolytic enzymes such as matrix metal- Serine proteinase from venoms of B. atrox and B. brazili were loproteinases have been implicated in modulating angiogenesis isolated using chromatographic techniques. A total of 150 mg of the (Pepper et al., 1996). These enzymes degrade extracellular matrix crude venoms of B. brazili and B. atrox were separately dissolved in (ECM) proteins to facilitate remodeling of ECM leading to release of 1.5 mL of a 0.02 M TriseHCl buffer pH 8.0 containing 0.15 M NaCl matrix bound growth factors thereby stimulating endothelial cell and were centrifuged at 10,000 Â g for 10 min. The clear super- migration and proliferation (Pepper, 2001; Sottile, 2004). Inter- natant was subjected to molecular size exclusion chromatography estingly, multiple functions of serine proteinases akin to thrombin, on a Sephacryl S-200 column previously equilibrated with the urokinase plasminogen activator, tissue plasminogen activator, aforementioned buffer. The proteins were eluted at a flow rate of kallikreins, tryptase and chymase have been demonstrated 0.2 mL/min, the absorption was monitored at 280 nm and fractions including induction of angiogenesis apart from their key role in of 1 mL each were collected. facilitating the coagulation cascade (Tsopanoglou and Maragoudakis, 2004). This prompted us to investigate whether 2.3.2. Affinity chromatography on benzamidine sepharose 4 Fast serine proteinases from Bothrops snake venoms have any influence Flow on modulating angiogenesis. In this study, we have extensively The fractions corresponding to the peaks containing serine characterized serine proteinases from B. brazili and B. atrox for their proteinases were pooled and then applied to a benzamidine molecular and functional properties and subsequently demonstrate sepharose 4 fast flow (high sub) column (5 mL bed volume) using a that serine proteinases exert robust pro-angiogenic activities superloop (50 mL) at a flow rate of 0.5 mL/min. The unbound in vitro and in vivo systems. proteins were washed out with 0.02 M Tris-HCl, pH 8.0, 0.15 M NaCl (buffer A). The nonspecifically bound proteins were eluted with the above mentioned buffer, which additionally contained 0.5 M NaCl. 2. Materials and methods Once the baseline was stabilized, the bound proteins were eluted by rapidly changing the pH to 3.0 using a 0.05 M glycine-HCl (buffer 2.1.